tbuckley/cabot-search huggingface dataset:
P12.08.A CELL-EXTRINSIC D-2-HYDROXYGLUTARATE PROMOTES HYPERMETHYLATION AT LINEAGE-SPECIFIC REGULATORY REGIONS TO PREVENT MICROGLIAL ACTIVATION IN IDH-MUTANT GLIOMAS
Authors: A Laurenge, P Pugliese, Q Richard, A Picca, S Jouannet, K Labreche, F Bielle, A Iavarone, M Ceccarelli, M Mallat, M Sanson, L Castro-Vega
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in diffuse gliomas. Their composition and transcriptional programs differ according to the IDH mutation status of glioma cells. The underlying mechanisms remain until now little known. MATERIAL AND METHODS We compared bulk DNA methylome (Methylation EPIC array) and transcriptome of TAMs (CD11b+ purified by magnetic-activated cell sorting) from 25 IDH-mutant and 11 IDH-wildtype gliomas. The contamination of the CD11b+ fraction by tumor cells was determined by the detection of IDH and/or TERT mutations using droplet digital PCR. Only samples with a purity of at least 95% were used for analyses. To determine experimentally direct effects of D-2-hydroxyglutarate (D-2-HG), which is the oncometabolite produced and realeased into the microenvironment by IDH-mutated glioma cells, we used primary cultures of human microglial cells obtained from glioma surgery or epilepsy surgery. We exposed microglia or not to (D-2-HG) during 14 days, and analyzed their DNA methylome and transcriptome. RESULTS TAMs from IDH-mutant gliomas compared with TAMs from IDH-wildtype gliomas exhibit decreased expression of pro-inflammatory pathways (mainly IFN-γ, IFN-α, and TNF-α signaling) and genes related to G2-M checkpoint, glycolysis and Epithelial-Mesenchymal Transition (EMT). We uncovered that TAMs from IDH-mutant gliomas exhibit significant hypermethylation at promoters and enhancers linked to decreased expression of genes involved in inflammatory responses. Notably, these hypermethylated regions were enriched for binding motifs of transcription factors controlling an environment-dependent transcriptional network in microglial cells (such as PU.1, AP-1, C/EBP and RUNX). These findings were confirmed by comparing methylome data of TAMs from IDH-mutant gliomas and methylome of microglia from normal brains available in the literature. A 14 days exposition to D-2-HG of human microglial cells in vitro partially recapitulates the observed methylation changes. Transcriptome analyses of these cells show that this oncometabolite dampens the IFN-α/γ pathways and attenuates the response to LPS while inducing a metabolic shift toward oxidative phosphorylation. CONCLUSION Our findings establish a link between an extrinsic cell-effect of D-2-HG and the hypermethylation of DNA of microglial cells at regulatory regions in IDH-mutant gliomas. Our observations support the concept that oncometabolites may disrupt directly the function of immune cells of the tumor microenvironment.
P20.10.B CHARACTERIZATION OF NEW PROGNOSTIC MARKERS IN A COHORT OF ADULT SPINAL EPENDYMOMAS
Authors: M Patanè, C Calatozzolo, L Maddaloni, R Paterra, G Marucci, M Eoli, M Farinotti, P Ferroli, A Silvani, B Pollo
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Spinal ependymomas (SP-EPN) are group of primary central nervous system tumors recently re-classified by the WHO (CNS5 2021), located in spinal cord. SP-EPN are grade 2-3 tumors, found mainly in cervical and thoracic region, characterized mostly by NF2 mutations and classical histology: grade 3 tumors present also microvascular proliferation, necrosis and high mitotic rate. Recently, a novel subtype was discovered with a unique methylation profile (SP-EPN-MYCN) and characterized by the amplification of MYCN gene, grade 3 histology and a very aggressive behavior. Treatment of these tumors remains challenging: gross-total resection is not often achieved and radiation failed to give sacceptable results. Besides, no prognostic factors except the mitotic index and tumor location, are available, thus, new diagnostic and prognostic biomarkers would be very useful for their accurate classification and management. MATERIAL AND METHODS We have collected 170 patients operated at our Institute from 1997 to 2019 of spinal ependymomas located in the cervical and thoracic region, excluded conus/filum mixopapillary ones. All tumors were revised according to the new WHO CNS5 2021 classification. In a cohort of 50 grade 2/3 SP-EPN we have evaluated by immunohistochemistry the expression of MYCN, H3K27me3, CDK4 and EZH2: the latter is a component of the PRC2 complex, which regulates the histone H3 methylation at Lys27 (K27) and recently described as a transcription factor which controls the expression of MYCN and CDK4 genes. We have also evaluated the amplification of MYCN and CDK4 genes by Copy Number Variation Assay and MLPA techniques. Clinical data were collected and related to the results obtained. RESULTS We have found strong overexpression of MYCN only in grade 3 SP-EPN (3/50), while grade 2 tumors showed low or no expression; amplification of MYCN gene was similarly confirmed by CNV assay and MLPA only in grade 3. Interestingly, also CDK4 overexpression was found only in grade 3 ependymomas (1/50), but it is expressed oppositely to MYCN: in tumors were MYCN is overexpressed, CDK4 was completely absent. Amplification of CDK4 gene was confirmed only in those with CDK4 overexpression. These results were both related to an interesting overexpression of EZH2 only in grade 3 SP-EPN (4/50), while H3K27me3 is expressed in all grades without differences. Finally, in our study both MYCN and CDK4-amplified ependymomas were related to a very aggressive behaviour and poor outcome with high recurrence rate, dissemination and metastases. CONCLUSION In this study we confirmed the prognostic role of MYCN as indicated by WHO classification and we have showed that CDK4 and EZH2 overexpression could be considered as new promising prognostic and predictive factors in SP-EPN. Since many trials with EZH2 and CDK4 inhibitors have been set up in the last years, these analyses could suggest a new therapeutic approach.
P16.09.B NEUROGENIC DYSPHAGIA IN MENINGEOSIS NEOPLASTICA PATIENTS
Authors: S Lapa, E Harborth, C Manß, M Ronellenfitsch, J Steinbach, H Urban
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Meningeosis neoplastica (MN) occurs in 5-10 % of all cancers and is characterized by diffuse seeding of tumor cells in the cerebrospinal fluid (CSF) and meninges. Common clinical symptoms suggestive of MN include headache, cranial nerve (CN) deficits, psychiatric symptoms as well as paresthesia. Neurogenic dysphagia (ND) has been suggested a rare MN symptom resulting from affection of CN IX and X. The aim of this work was to investigate frequency, severity and pattern of malfunction of ND due to MN as well as to assess its impact on the rate of pneumonia and overall survival. MATERIAL AND METHODS The database of the Institute of Neurooncology of the University Hospital Frankfurt was screened for patients with MN diagnosed between March 2009 and July 2021 who received a systematic dysphagia assessment (DA). DA comprised a standardized clinical swallowing examination as well as an additional fiber optic endoscopic evaluation of the swallowing act (FEES) in case of suspected aspiration. Severity of dysphagia and extent of oral impairment were systematically recorded. In addition to demographics, clinical diagnoses, underlying cancer and treatment, information on radiological imaging including presence of contrast-enhancing lesions was collected. RESULTS A total of 262 patients with a diagnosis of MN were detected, 69 of whom (52 % women, mean age 61 ± 14 years) were included for further analysis. Underlying cancers included lung cancer (16 cases, 23 %), breast cancer (15, 22 %), as well as lymphoma and melanoma (each 7 cases, 10%). The remaining 24 patients suffered from cancer entities (ovarian cancer, gastrointestinal tumors or cancers of unknown primary). 46 patients (66.6 %) were diagnosed with ND, 15 of these patients required a feeding tube (FOIS 1-3). Dysphagic patients had an increased risk of pneumonia (p&amp;lt;0.001) and a shorter survival time compared to non-dysphagic patients (p=0.003). Affection of the cranial nerves affected CN V (n=6), CN VII (n=22), CN IX (n=21), CN X (n=7) and CN XII (n=12). There was no significant correlation between the presence of an ND and the above-mentioned cranial nerve deficits. CONCLUSION The results of this study show that ND is a common symptom in patients with MN and can also occur independently of paresis of CN IX and X. In addition, the presence of ND is associated with a higher rate of pneumonia and a shorter survival time. Therefore, patients with suspected or confirmed MN should receive a systematic dysphagia assessment. In addition, the presence of MN should also be considered in the differential diagnosis of unclear neurogenic dysphagia.
P12.10.A VASCULOGENIC MIMICRY OCCURS IN PRIMARY AND RECURRENT GLIOBLASTOMA AND IS NOT ASSOCIATED WITH PSMA EXPRESSION
Authors: P Tooney, K Maddison, S Faulkner, N Bowden, M Graves, M Fay
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Glioblastomas are highly vascular tumours that overexpress vascular endothelial growth factor (VEGF), however the anti-VEGF antibody bevacizumab has not demonstrated significant overall survival benefit for patients. Vasculogenic mimicry (VM), the ability of tumour cells to form functional microvasculature lacking an endothelial lining, may compensate for insufficient or inhibited angiogenesis. VM has been reported in primary glioblastoma tissue, but not investigated at progression in recurrent tumours. Prostate specific membrane antigen (PSMA) is expressed in abnormal vessels within primary glioblastoma tumours. We aimed to determine whether VM occurs in primary and recurrent glioblastoma tumours and whether VM vessels also express PSMA. MATERIAL AND METHODS Thirty-five matched pairs of primary and recurrent formalin-fixed paraffin-embedded glioblastoma tissue were immunohistochemically labelled for the endothelial cell marker CD34 and PSMA, and counterstained with periodic acid-Schiff (PAS). Vascular structures were categorised as endothelial vessels (CD34+/PAS+) or VM (CD34-/PAS+). Vessels were counted in ten regions within each tumour and a vessel density/mm2 for each vessel category was determined. RESULTS Data are expressed as median values. Endothelial vessels accounted for most blood vessels observed. There was a significant decrease in endothelial vessel density in recurrent (41.34 vessels/mm2) compared to primary (86.98 vessels/mm2) glioblastomas (p &amp;lt;0.001). VM was observed in 15/35 primary tumours (42.86%) and 10/35 recurrent tumours (28.57%). VM accounted for only a small proportion of the overall vasculature and VM density was not different between the primary and recurrent groups (p = 0.266). Kaplan-Meier analyses with log rank tests indicated that VM-tumours had longer median OS time than VM+ tumours, regardless of whether VM status was determined based on the primary or recurrent tumour. However, there was no significant difference in survival distribution between VM+ and VM-tumours (primary χ2(1) = 0.381, p = 0.537; recurrent χ2(1) = 3.552, p = 0.059). PSMA is expressed in endothelia lined vessels based on serial section labelling for PSMA and CD34. PSMA expression significantly decreased in recurrent (median H-score = 0.22) compared to primary (median H-score = 0.44) tumours (one-tailed p = 0.031; n = 32) mirroring the CD34+ vessel density. CONCLUSION VM contributes to a relatively small proportion of the overall vasculature in glioblastoma tumours and was not associated with OS nor the expression of PMSA. Since VM- tumours had longer OS than VM+ tumours, assessment of VM in recurrent tumours in a larger cohort may determine if there is a clinical significance for VM in recurrent glioblastoma.
P19.03.B IMPLEMENTATION OF RESECT CATEGORIES FOR EXTENT OF RESECTION STRESS THE LONG-TERM CLINICAL BENEFIT OF SUPRAMAXIMAL CONTRAST ENHANCING RESECTION IN A RETROSPECTIVE COHORT OF NEWLY DIAGNOSED IDH WT GLIOBLASTOMA PATIENTS
Authors: C Gambacciani, M G Nizzola, F Calvanese, F Villanacci, F Pieri, G Grimod, F Aquila, C Scudieri, S Cupini, I Stasi, V Zucchi, A Giusti, L Lastrucci, F Vannozzi, A Giuri, L Farina, A Pichiecchio, A L Di Stefano, O S Santonocito
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND to assess the long-term benefit associated with supramarginal CE resection and to apply ReSECT criteria for extent of resection in patients with primary glioblastoma IDH wild-type. MATERIAL AND METHODS We performed a retrospective search in the institutional of Livorno Hospital for adult patients with primary GBM IDH wild-type. We assess residual tumor volumetry in the early post-operative magnetic resonance imaging by the 3dslicer software. We attributed EOR classes according to ReSECT criteria based on residual contrast and non-contrast enhancing tumor volumes. We performed multivariate analysis by Cox regression including main known prognostic factors and MGMT promoter status assessed by pyrosequencing. IDH wild-type status was defined on both IDH1 and IDH2 genes. RESULTS We identified 173 patients who underwent surgery for confirmed primary glioblastoma IDH wild-type in our institutions from 2016 and 2022. Male/female ratio was 1.6, median age at diagnosis 68 years old (yo) (range 30-83), median KPS before surgery was 80 (range 30-100) with no significant variation at the post-operative stage. Forty out of 173 (23%) patients received a supramarginal CE resection (class 1) in the whole cohort; prevalence of class 1 resection was the highest in the subgroup of patients aged ≤ 65 yo (27%) than the subgroup of &amp;gt;65 ≤ 75 yo (22%) and the subgroup of &amp;gt;75 yo (14%). The rate of class 1 resection was not different between patients with tumors located in the right and in left hemisphere (24% and 23% respectively). We analysed survival in patients treated with adjuvant RT-TMZ and found a significant longer OS in patients operated with resection class 1 (16.7 months) than other classes (p&amp;lt;0.0001). Long term benefit resulted the strongest in the subgroup of patients aged ≤ 65 yo (median OS 23.8 months). Multivariate analysis confirmed EOR according to the ReSECT criteria as the strongest independent predictor factor of OS (HR 0.6, p=0.004). CONCLUSION We efficiently implemented ReSECT criteria for EOR in our retrospective cohort of patients with primary GBM IDH wild-type. Supramarginal CE resection defined as no residual CE and ≤ 5 cm of nCE on the post-operative imaging emerged as the strongest predictive factor of survival.
P02.13.A THIRD-GENERATION SEQUENCING OF BRAIN TUMORS ON SINGLE-CELL LEVEL
Authors: N Schoebe, F M Ippen, M Ritter, Y Tang, W Wick, A von Deimling, F Sahm, N Berghaus
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Intratumor heterogeneity (ITH) in brain tumors has been suggested to increase tumor adaptability and thus drive tumor progression and treatment resistance. Single cell sequencing has widened the range of possibilities in detecting clonal diversity and tumor evolution. As of now, most single cell experiments rely on next-generation sequencing (NGS) platforms. However, they detect only short RNA/DNA fragments that do not show the full picture of existing transcript isoforms or genetic variants. New, long-read sequencing methods such as Oxford Nanopore Technologies (ONT) or PacBio sequencing may now enable novel approaches to single cell experiments. We here set-out to complement existing single-cell analysis workflows and data pipelines with long-read sequencing approaches. We compare and integrate the data output in order to advance the understanding of ITH in brain tumors, paradigmatically demonstrated on meningioma. MATERIAL AND METHODS We set up a pipeline for single-cell/nuclei RNA long-read sequencing on ONT devices. First, libraries as derived from the 10x genomics workflow were employed, subsequently followed by independently generated libraries. To widen the insight into ITH on multiple levels, we next assessed single-cell/nuclei DNA for small somatic variants and methylation along a modified version of this platform. RESULTS Long-read RNA-sequencing yielded results comparable to short-read sequencing. Depending on sample quality, higher reads per cell and library were required for ONT sequencing to achieve similar discrimination of clusters by expression. On the other hand, cell-type specific isoforms could be identified. Specific small somatic variants known from bulk sequencing data were identified when the region was sufficiently covered in the RNA data, which was naturally more often achieved in long read than short-read data. Thus, cell-specific variant calling was enabled in a high-throughput pipeline. Maintaining the methylation information in DNA sequencing could so far only be set-up in a plate-based approach. CONCLUSION Using these approaches, we resolved features of ITH and clonal tumor evolution, aiding us in gaining a broader understanding of brain tumor biology. Furthermore, it showed novel approaches that could possibly be implemented in routine diagnostic procedures in the future. These novel techniques could be applied to other tumor entities and possibly further omics layers such as single cell proteomics and metabolomics could be added to gain a greater understanding of different tumor types and their clonal diversity.
P10.17.A GLUTAMINE METABOLISM AS A PROGNOSTIC FACTOR IN GBM AND OLIGODENDROGLIOMA
Authors: M Trybula, M Lysiak, A Malmstrom, P Soderkvist
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND Increased uptake of principal nutrients is one of the attributes of cancer cells. A recent study has shown that glutamine, but not glucose, consumption differed markedly between gliomas and adjacent healthy tissue. However, the prognostic role of glutaminolysis or glutaminolysis-related genes (GRGs) in gliomas remains unclear. Here we sought to determine if the GRG signature correlates with glioma patient survival and if it could identify patients with different outcomes. MATERIAL AND METHODS The RNA-seq and clinical data for The Cancer Genome Atlas (TCGA) was obtained from the GlioVis webpage. We compared the expression of the GRGs (GLUD1, GLUL, GLS1, GLS2, GOT1, GOT2, GPT, GPT2, SLC1A5, and SLC7A5) between 4 non-tumor, 220 IDH wild-type (IDHwt) and 389 IDH mutant (IDHmut) gliomas, of which 154 harbored 1p/19q codeletion. We further performed unsupervised hierarchical clustering for all glioma patients and compared survival between clusters. RESULTS Our results revealed that most GRGs were differentially expressed between non-tumor, IDHwt, and IDHmut gliomas. We found that gliomas grouped into 3 clusters based on the varying GRGs expression. Eighty-eight percent of glioblastomas (GBM, IDHwt) and 19% of all IDHmut tumors were characterized by low GRGs expression. These were denoted as cluster 1, and the remaining samples belonged to clusters 2 or 3, corresponding to intermediate and high GRGs expression groups, respectively. We performed survival analysis separately for IDHwt and IDHmut gliomas and found that cluster 1 patients had the worst prognosis, regardless of IDH status. We further separated IDHmut gliomas according to 1p/19q codeletion status. Oligodendroglioma (1p/19q codeleted) patients had a median survival of 33.2 months, 134.3 months, and 169.8 months in clusters 1, 2, and 3, respectively. No cluster-dependent differences in survival were seen for astrocytoma. For IDHwt (GBM) we found that patients from clusters 1 and 2 had survival of 14 months and 25 months, respectively. Due to the small number of deceased GBM patients in cluster 3, we could not estimate the median survival. The univariate analysis confirmed that cluster membership was an independent predictor of survival in oligodendroglioma (cluster 2 and 3 vs. 1, HR 0.14; 95% CI, 0.05-0.4; p=0.0005) and GBM (cluster 1 vs. 2 and 3, HR 3.05; 95% CI, 1.5-5.9; p=0.0012). CONCLUSION The GRG signature could define metabolic groups with distinct survivals in oligodendroglioma and GBM and constitute a new prognostic factor. We are planning further investigations to confirm our findings.
P11.60.A RACTAC TRIAL: PHASE II MULTICENTRIC ITALIAN TRIAL ON REPOSITIONING OF THE ANTIPSYCHOTIC DRUG CHLORPROMAZINE AND ITS COMBINATION WITH TEMOZOLOMIDE IN MGMT UNMETHYLATED GLIOBLASTOMA PATIENTS
Authors: A Pace, G Lombardi, V Villani, D Benincasa, C Abruzzese, I Cestonaro, M Corrà, G Cerretti, M Caccese, A Silvani, P Gaviani, D Giannarelli, M G Paggi
Journal: Neuro-Oncology (2023)

Abstract BACKGROUND The poor prognosis of patients affected by glioblastoma (GBM) prompts the search for new and more effective therapies, particularly for GBMs with unmethylated MGMT. In this regard, drug repurposing, can represent a safe and inexpensive way to bring novel pharmacological approaches from bench to bedside. Chlorpromazine, a medication in use since six decades for the therapy of psychiatric disorders, shows in vitro features that make it eligible for repositioning in GBM therapy. In our experimentation on six GBM cell lines, chlorpromazine inhibited cell viability in an apoptosis-independent way, induced polyploidy, reduced cloning efficiency as well as neurosphere formation and downregulated the expression of stemness genes. Notably, we found that chlorpromazine synergized with temozolomide, in reducing cell viability and strongly cooperated in reducing cloning efficiency and inducing cell death in vitro for all the GBM cell lines assayed. MATERIAL AND METHODS With these assumptions, we started a multicentric Phase II clinical trial on newly diagnosed GBM patients with unmethylated MGMT by adding chlorpromazine to temozolomide in the adjuvant phase of the standard first-line therapeutic protocol. The experimental procedure involves the combination of CPZ with standard treatment with TMZ in the adjuvant phase of the Stupp protocol. CPZ was administered orally at a dose of 50 mg/day - GG 1-28 - of every cycle of the adjuvant treatment with TMZ. . Efficacy outcomes were evaluated using Kaplan-Meier methodology. OS was measured from diagnosis to death; PFS was determined from radiotherapy to the first sign of PD or death due to any cause. RESULTS The trial was closed on December 2022. 53 patients have been enrolled, 39 male, 14 female. 21 patients completed 6 cycles of treatment, without relevant toxicity.The results shows a median PFS of 7 months (St.Dev. 5,7) and a median OS of 15 months (St.Dev. 6,9). 23 patients are still alive (43%) CONCLUSION The addition of Chlorpromazine to the standard adjuvant chemotherapy with temozolomide was well tolerated in newly diagnosed Glioblastoma unmethylated patients, with promising impact on outcome measures. On the basis of the RACTAC trial RESULTS , a phase II multicentric trial evaluating Chlorpromazine added to standard treatment (RT+TMZ+adj TMZx 6 cycles plus CPZ) in newly diagnosed Glioblastoma patients is in preparation